C16, a PKR inhibitor, suppresses cell proliferation by regulating the cell cycle via p21 in colorectal cancer

Double-stranded RNA-activated protein kinase R (PKR) is highly expressed in colorectal cancer (CRC). However, the role of PKR in CRC remains unclear. The aim of this study was to clarify whether C16 (a PKR inhibitor) exhibits antitumor effects and to identify its target pathway in CRC. We evaluated the effects of C16 on CRC cell lines using the MTS assay. Enrichment analysis was performed to identify the target pathway of C16. The cell cycle was analyzed using flow cytometry. Finally, we used immunohistochemistry to examine human CRC specimens. C16 suppressed the proliferation of CRC cells. Gene Ontology (GO) analysis revealed that the cell cycle-related GO category was substantially enriched in CRC cells treated with C16. C16 treatment resulted in G1 arrest and increased p21 protein and mRNA expression. Moreover, p21 expression was associated with CRC development as observed using immunohistochemical analysis of human CRC tissues. C16 upregulates p21 expression in CRC cells to regulate cell cycle and suppress tumor growth. Thus, PKR inhibitors may serve as a new treatment option for patients with CRC.


C16 suppresses the proliferation of CRC cells in a dose-dependent manner in vitro
The MTS assay was performed to assess the proliferation of CRC cells treated with 0, 100, 500, and 1000 nM C16.The division of HCT116 cells decreased following C16 treatment in a dose-dependent manner (Fig. 2a).The suppressive effect of C16 treatment on CRC cell proliferation was also observed in another p53 mutant CRC cell line, HT29 (Fig. 2b).No morphological changes were observed in the cells 24 h after C16 exposure, suggesting that this inhibitor does not exert substantial toxic effects (Fig. 2c).
Figure 1.PKR induces the proliferation of CRC cells.HCT116 cells were seeded in a six-well flat-bottomed plate and transfected with PKR plasmid and PKR siRNA1 or siRNA2 for 48 h; thereafter, proteins were extracted and analyzed.The expression of PKR in HCT116 was determined using western blotting.The original gel images of western blotting are shown in Supplemental Fig. S1 (a).To investigate the effects on CRC cell proliferation in vitro with the MTS assay, CRC cells were seeded in a 96-well flat-bottomed plate with the PKR plasmid and PKR siRNA.Data are shown as mean ± standard error of six replicates.*p < 0.05, **p < 0.01 compared to the group with control plasmid and control siRNA using Mann-Whitney U test (b).PKR protein kinase R, CRC colorectal cancer.

C16 regulates CRC cell cycle
Flow cytometry showed that C16 regulates CRC cell cycle.Following treatment with C16, the number of HCT116 cells in the G1 phase increased and the number of cells in the S and M phases decreased (Fig. 4a).The regulatory effects of C16 treatment on CRC cells were also observed in HT29 cells (Fig. 4b).These results demonstrate that C16 regulates the cell cycle by inhibiting the transition from the G1 to S phase.In addition, apoptosis assays using flow cytometry showed that the rate of apoptosis clearly increased after 48 h C16 treatment.The percentage of annexin V-positive cells defined as apoptotic cells was as follows: control DMSO, 10.5%; C16, 19.4% in HCT116 cells (Supplemental Fig. S2).www.nature.com/scientificreports/

C16 increases p21 expression in CRC cells
We performed western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments to examine the effects of C16 on p21 expression after exposing HCT116 cells to 0, 100, 300, or 500 nM of C16.C16 increased p21 and p53 protein expression in HCT116 and HT29 cells (Fig. 5a).Furthermore, it increased CDKN1A and TP53 mRNA expression in HCT116 cells (Fig. 5b) and CDKN1A expression in HT29 cells (Fig. 5c).These results indicate that C16 regulates p21 expression.We also performed western blotting and MTS assay to confirm that p21 siRNA reverses the tumor growth-suppressive effects of C16.p21 protein expression was knocked down by p21 siRNA1 and siRNA2 (Fig. 5d).We subsequently transfected p21 siRNA to HCT116 cells treated with C16.The MTS assay showed that p21 siRNA reverses the tumor growth-suppressive effects of C16 (Fig. 5e).These results indicate that the tumor growth-suppressive effects of C16 is dependent on p21.

p21 expression decreases in advanced human CRC tissues
We analyzed tumor tissues from patients with CRC to investigate the role of p21 in CRC development in vivo.Fisher's exact test was used to examine the correlation between p21 expression and clinicopathological characteristics.The clinicopathological characteristics of patients with CRC are summarized in Table 1.p21 expression was low in 55% (12/22) and high in 45% (10/22) of the CRC tissues (Fig. 6a).Immunohistochemical (IHC) analysis revealed that low p21 expression positively correlated with the tumor, node, metastasis stage (p = 0.043) and tumor diameter (p = 0.048) (Fig. 6b, c).In contrast, there was no association between p21 expression and age, sex, or tumor location.These results suggest that p21 expression is associated with CRC development.

Discussion
This study investigated the effects of the PKR inhibitor C16 on CRC cells.C16 increased p21 expression and p21 upregulation induced G1 arrest and suppressed CRC cell proliferation.
Although there are several reports on the therapeutic effects of PKR inhibitors in neurological diseases [20][21][22] , there are only a few reports on the effects of PKR inhibitors on malignant tumors.Our previous study showed that C16 treatment suppresses tumor progression by suppressing the proliferation of tumor cells and the expression of angiogenesis-related growth factors, such as vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factors, and epidermal growth factor in HCC 19 .Furthermore, C16 induces apoptosis in a dose-dependent manner and suppresses CRC cell proliferation 23 , decreases p-eIF2α level, and induces MYC expression in patient-derived CRC organoid lines.However, previous studies did not demonstrate that C16 inhibits CRC cell proliferation via the cell cycle.In addition, MYC overexpression induces the progression of cell cycle 24,25 and inhibits the induction of p21 expression 26,27 .Our results reveal that different pathways are involved in the effects of C16 treatment.To our knowledge, this is the first study to show that the inhibitory effect of C16 treatment on tumor growth is regulated by the CRC cell cycle.
We found that C16 suppressed the proliferation in the wild-type p53 CRC cells HCT116 and p53-mutated CRC cells HT29.Furthermore, C16 regulated the expression of cell cycle-related genes and increased p21 protein and mRNA expression independent of TP53 mutations.The western blot analysis showed that C16 did not affect the protein expression of p53 or phosphorylated p53, whereas the RT-PCR analysis showed that C16 increased the mRNA expression of TP53 in HCT116 cells (wild-type TP53) but did not affect the mRNA expression of TP53 in HT29 cells (mutant type of TP53).Furthermore, the enrichment analysis showed that C16 affected the expression of cell cycle-related genes including TGF-β-related genes.Together, these findings suggest that an alternative pathway (such as the TGF-β pathway rather than the p53 pathway) is the key pathway by which C16 regulates the cell cycle.
ATP competitive inhibitors such as C16 have been reported to induce integrated stress responses depending on their concentration 28 .We conducted this study with C16 at a low concentration that is less likely to induce integrated stress responses (ISR).Furthermore, PKR inhibition by PKR knockdown suppressed cell proliferation, and cell proliferation inhibition by C16 was negated by p21 inhibition.From these results, it cannot be ascertained that C16 induced ISR.Further investigation is needed to elucidate the relationship with ISR.
Our analysis of human CRC tissues showed that p21 expression is related to CRC development.The expression of p21 decreased as tumor tissue atypicality increased.The expression of p21 mRNA is lower in CRC tissues than in normal colorectal tissues according to a public database.Furthermore, a previous IHC study showed that p21 expression is inversely related to Ki67 proliferation and that it could be related to p53 inactivation 29,30 .Loss of p21 is associated with a poor prognosis in CRC 31 .In vitro and in vivo experiments demonstrate that p21 plays a role in tumor growth suppression in CRC cells 9,32,33 .In addition, loss of CDKN1A (p21) is associated with consensus molecular subtype (CMS) 4 CRC, which has worse relapse-free and overall survival rates compared to CMS 1-3 34 .Therefore, C16 may improve cancer prognosis by restoring p21 expression.
Several studies have investigated the effects of drugs and chemicals on p21 expression in cancer cells.For example, histone deacetylase inhibitors (trichostatin A, bortezomib, or doxorubicin) enhance p21 expression and induce cell cycle arrest 35,36 , demonstrating the tumor-suppressive effects of p21.However, there are contradictory reports on the tumor growth effects of p21 37,38 .p21 loss in colon cancer is associated with shorter survival among patients aged < 60 years, whereas it is associated with longer survival among patients aged ≥ 60 years 39 .p21 plays tumor-suppressive and tumor-proliferative roles; however, the development of clinically available p21-targeted therapeutics for CRC has not yet been achieved.
In conclusion, we found that C16 treatment inhibits tumor growth by regulating CRC cell cycle.Thus, PKR inhibition could be a novel p21-targeting therapy to regulate CRC.

Cell lines and cell culture
Human CRC cell lines HCT116 and HT29 were purchased from KAC (Kyoto, Japan).HCT116 and HT29 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin.The cells were maintained at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air, and the culture medium was changed three times per week.www.nature.com/scientificreports/

PKR plasmids
Plasmids carrying PKR genes were kindly provided by Dr. Michael Gale, Jr. (University of Washington, Seattle, WA, USA).The plasmid pOS8, which expresses β-galactosidase, was used as the control plasmid.Each plasmid (1 mg/mL) was transfected into HCT116 at 50% confluence in six-well plates using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific).

Chemicals
C16 was purchased from Merck (Darmstadt, Germany) and solubilized in dimethyl sulfoxide (DMSO).The final concentrations of PKR inhibitors used in the in vitro experiments are described in the following sections.

Cell proliferation assay
In vitro cell viability was quantified using the MTS assay (Promega, Fitchburg, WI, USA).After 24 h of siRNA or plasmid transfection in six-well plates, the cells were resuspended and seeded at a density of 2 × 10 3 in 96-well plates.HCT116 and HT29 cells treated with C16 (Merck) at various concentrations (100-2000 nM) were seeded in 96-well plates at 2 × 10 3 cells per well.The cells were treated with MTS reagent and incubated for 120 min.
Absorbance was recorded at 450 nm.Cells treated with DMSO were used as controls.The wells were photographed under a microscope (ZEISS Axio Vert.A1; Carl Zeiss, Oberkochen, Germany).

RNA-Seq and data analysis
Total RNA was collected from HCT116 cells 24 h after treatment with control DMSO or C16 diluted to 500 nM, and the integrity of the isolated RNA was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).RNA samples with RNA integrity number > 9 were normalized to 100 ng/μL before further analysis.RNA-Seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; NEB, Ipswich, MA, USA), NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), and NEBNext Multiplex Oligos for Illumina (NEB) according to the manufacturer's instructions and subsequently validated to ensure an average size of approximately 380-390 bp using the Agilent 2100 Bioanalyzer and the Agilent DNA1000 Kit (Agilent Technologies).Paired-end reads (75 bp) were sequenced using the MiSeq Reagent Kit V3 (Illumina, San Diego, CA, USA) on a MiSeq system (Illumina).Mapping to human genome data of hg38 was performed using Tophat (https:// ccb.jhu.edu/ softw are/ tophat/ index.shtml), and the expression analysis tool Cufflinks (http:// cole-trapn ell-lab.github.io/ cuffl inks/) was used to normalize expression in each sample before determining the differences in expression between the control DMSO (n = 3) and C16 (n = 3).Pathway and process enrichment analyses were performed using all genes after alignment and normalization with adjusted p < 0.05 using the Metascape and DAVID analysis tools 40,41 .

Cell cycle analysis
HCT116 and HT29 cells were seeded at a density of 1 × 10 6 cells/well in 10-cm dishes.A BD Cycletest™ Plus DNA Kit (BD Biosciences, Franklin Lakes, NJ, USA) was used to purify the samples at 24 h after treatment with C16 diluted to 500 nM, according to the manufacturer's instructions, and cell cycle distribution was evaluated using flow cytometry (Gallios Flow Cytometer; Beckman Coulter, Brea, CA, USA).

Apoptosis assay
HCT116 cells were treated with control DMSO or C16 diluted to 500 nM for 48 h.The attached cells were collected and assayed to evaluate apoptosis with annexin V-PE and 7-amino-actinomycin D (Tonbo Biosciences, San Diego, CA).To visualize stained cells, we used a FACSCalibur system (Becton Dickinson, Franklin Lakes, NJ).The data were analyzed with the FlowJo software (TreeStar Corporation, Ashland, OR).

Figure 2 .
Figure 2. Treatment with C16 PKR inhibitor suppresses CRC cell proliferation in a dose-dependent manner in vitro.(a) HCT116 cells, (b) HT29 cells.Data are shown as mean ± standard error of six replicates.*p < 0.05, **p < 0.01 compared to the group treated with DMSO (Mann-Whitney U test).(c) HCT116 cells were seeded in a six-well plate, cultured at 37 °C for 24 h, and treated with C16 at 100, 500, 1000, and 2000 nM.The microscopic photographs of the wells with C16 at different concentrations.Scale bar, 200 µm.DMSO dimethyl sulfoxide, PKR protein kinase R, CRC colorectal cancer.

Figure 3 .
Figure 3. C16 regulates the expression of cell cycle-related genes in CRC cells.Functional enrichment analysis was performed using Metascape and DAVID.RNA-Seq analysis was performed using RNA extracted from HCT116 cells 24 h after treatment with C16 diluted to 500 nM or DMSO (control).Functional enrichment results for GO (a) and KEGG (b) pathways, and a heatmap showing the top 19 and 20 clusters colored according to the p-value (a darker color indicates a lower p-value).(c) RNA-Seq analysis of cell cycle-related gene expression.The y-axis shows the ratio of the average value of fragments per kilobase of transcript per million mapped reads (FPKM) of three HCT116 cells treated with C16 to the average value of cells treated with DMSO.DMSO dimethyl sulfoxide, PKR protein kinase R, CRC colorectal cancer, GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes.

Figure 4 .
Figure 4. C16 regulates the cell cycle of CRC cells.Flow cytometry analysis was performed after treatment with C16 diluted to 500 nM.Two experimental replicates of cell cycle phase distribution were quantified.The number of cells in the G1 phase increased, whereas the number of cells in the S and G2 phases decreased.(a) HCT116 cells; (b) HT29 cells.**p < 0.01, *p < 0.05 compared to the group without C16 treatment (Mann-Whitney U test).DMSO dimethyl sulfoxide, PKR protein kinase R, CRC colorectal cancer.

7 Figure 5 .
Figure 5. C16 increases the expression of p21 in CRC cells.CRC cells were treated with 100, 300, and 500 nM C16 for 24 h.DMSO was used as a control.The expression of p53, phosphorylated p53, and p21 was determined using western blotting in HCT116 and HT29 cells.The original gel images of western blotting are shown in Supplemental Fig. S3 (a).The expression of TP53 and CDKN1A mRNA was measured using real-time PCR.(b) TP53, (c) CDKN1A.Data are shown as mean ± standard error of six replicates.**p < 0.01 compared to the group without C16 treatment (Mann-Whitney U test).HCT116 cells were seeded in a six-well flat-bottomed plate and transfected with p21 siRNA1 or siRNA2 for 48 h treatment, and then proteins were extracted and analyzed.The expression of p21 was determined using western blotting in HCT116.The original gel images of western blotting are shown in Supplemental Fig. S1 (d).To investigate the effects on CRC cell proliferation in vitro with the MTS assay, CRC cells were seeded in a 96-well flat-bottomed plate with C16 and p21 siRNA.Data are shown as mean ± standard error of six replicates.**p < 0.01 compared to the control group (DMSO) with Mann-Whitney U test (e).DMSO dimethyl sulfoxide, PKR protein kinase R, CRC colorectal cancer.

Figure 6 .
Figure 6.Low expression of p21 in advanced human CRC tissues.(a) Evaluation of p21 expression in human CRC tissues using immunohistochemical analysis.Data were generated from two representative CRC tissues for low and high p21 expression using the anti-p21 antibody.Scale bar: 100 μm.p21 expression in CRC tissue compared according to the TNM stage (b) and tumor size (c).Data are shown as mean ± standard error.*p < 0.05 compared to the high p21 expression group (Fisher's exact test).TNM tumor, node, and metastasis, CRC colorectal cancer.

Table 1 .
Relationship between p21 expression and clinicopathological features of patients with colorectal cancer (CRC).a Two-sided Fisher's exact test.